Rod transducin (Tr), a heterotrimeric GTP-binding protein composed of alpha, beta, and gamma subunits, couples photolysis of rhodopsin to the activation of cyclic GMP phosphodiesterase in the vertebrate visual signal transduction cascade. To determine if T alpha r is covalently modified, we analyzed tryptic fragments of bovine retinal T alpha r using electrospray mass spectrometry, liquid chromatography/mass spectrometry, tandem mass spectrometry, and gas chromatography. A novel heterogeneous fatty acylation was detected at the NH2 terminus. Four types of NH2-terminal tryptic fragments of T alpha r were isolated, and each contained either a lauroyl (C12:0), myristoyl (C14:0), (cis-delta 5)-tetradecaenoyl (C14:1) or (cis,cis-delta 5, delta 8)-tetradecadienoyl (C14:2) fatty acyl residue amide-linked to the NH2-terminal glycine residue. NH2-terminal fatty acylation does not anchor T alpha r permanently in the membrane, since T alpha r used in these experiments was eluted without detergent from rod outer segment membranes.